LOCUS       sequence-212241-        7427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA plasmid for cleavage of Candida albicans ADE2 - integrates at 
            RPS1 (Part of Duet system).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7427)
  AUTHORS   Vyas VK, Barrasa MI, Fink GR
  TITLE     A CRISPR system permits genetic engineering of essential genes and 
            gene families.
  JOURNAL   Sci Adv. 2015;1(3):e1500248.
  PUBMED    25977940
REFERENCE   2  (bases 1 to 7427)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2015"; volume: "1(3)"; pages: "e1500248"
COMMENT     Sequence Label: pV1010
FEATURES             Location/Qualifiers
     source          1..7427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      28..616
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     517..536
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     770..787
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    904..925
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(940..945,946..963,964..970)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    978..994
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     983..1005
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1002..1018
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1002..1018
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1037..1057
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1039..1057
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_RNA        4528..4603
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(5651..5670)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5652..5670)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5680..5697)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5680..5696)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5689..5711)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5838..6293
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5925..5944)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6135..6156
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6319..6423
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6424..6492,6493..7284)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6642..6661)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
       61 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
      121 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
      181 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
      241 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
      301 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
      361 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
      421 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
      481 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
      541 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
      601 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
      661 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
      721 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
      781 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
      841 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
      901 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgctcccggc
      961 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     1021 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg gtacctggtt
     1081 gttaagtcag tagatgattt gttgttgtcg tttgattttg ttacagcgta accagtgcgt
     1141 tttgtttgtt tccacatcat acacttcact gaaactaaat aagtttgttt acattttgag
     1201 acttcaggta cgacccaggg ttgcgacaaa gtttaggtag tttgtcgtct gaatgtcgca
     1261 acaaaatagg gctgtagccc tagtcatgtg atgtgaatta acagaacaag aagaactgct
     1321 ggtgcgcaaa aagattatgt gtattttatg tgcgttgtta tcctgcacac taaaattgag
     1381 cagtgtacac acacacatct tgggctgtat ttttattctt gtttttctgg tgttctctca
     1441 ctgttaagct ctaagtgaat ttgtgtgtgc tgtaatagtg tgtgtgttcc aagtcccagc
     1501 tctcacagat actcacgcac gcccatacta ctgaaaattt cctgactttc tgtatctaaa
     1561 aattttttac taggaatttt tttcttttac gtttttcact tgtttcatat aatcaccaac
     1621 tcaagtacaa cagatctgga ccacctttga ttgtaaatag taataattac cacccttatc
     1681 taattattta tttaacttat ttatttattt attatacata tatacaaatc taataaagtg
     1741 aaaatctccc ccttcacact tcacatatgt taggcgtcat cctgtgctcc cgagaaccag
     1801 taccagtaca tcgctgtttc gttcgagact tgaggtctag ttttatacgt gaagaggtca
     1861 atgccgccga gagtaaagcc acattttgcg tacaaattgc aggcaggtac attgttcgtt
     1921 tgtgtctcta atcgtatgcc aaggagctgt ctgcttagtg cccacttttt cgcaaattcg
     1981 atgagactgt gcgcgactcc tttgcctcgg tgcgtgtgcg acacaacaat gtgttcgata
     2041 gaggctagat cgttccatgt tgagttgagt tcaatcttcc cgacaagctc ttggtcgatg
     2101 aatgcgccat agcaagcaga gtcttcatca gagtcatcat ctgagatgta atccttccgg
     2161 taggggctca cacttctggt agatagttca aagccttggt cggataggtg cacatcgaac
     2221 acttcacgaa caatgaaatg gttctcagca tccaatgttt ccgccacctg ctcagggatc
     2281 accgaaattt tcatatgaga accgttatcg ataactaaag cagcaacttc ttctataaaa
     2341 atgggttagt atgacagtca tttaaataag gaatttttca gttggcttgg tttcaattca
     2401 atgttcgttt ttttttttct tgctgtgttt gtgtttgtgt tgtttatagt tgtgtgcact
     2461 gatcgtcgaa aaaaaaaatt catagtgagc cgggaaatct gtatagccca gataacaaca
     2521 caagtccaaa ctagaaactc gtcaaacacc aaaagcaatg ttgaatcaat tgccttgcac
     2581 aagtacacgt aggaaaacat aaaacattgc aattttgaat attgagcctt ttgtcgtaac
     2641 attgattgat aggattactc accgaatggt tttgaaacca ctgccgacag atcaatcaat
     2701 caatcaaaaa acgtgaactt tgaaaaaggg gaagaacaga tacattgaag ttagccattt
     2761 ccactgatcg tcacaacata tctgataaat tactttcaaa attataagct gatgtgtgtg
     2821 tattattaat gtgacagtaa catcccaaac gagaaatatt atctcgacaa caaaaaagtt
     2881 tgatctgaat tgaaaatgaa gttttcccac cctacccatt tgtcatattg aaaccaatca
     2941 actgattaat caatcaatta gaattgaagc taaactaaaa cataccaccg tccattttga
     3001 atgattatat ttttttaata ttaatatcga gataatgttt ctaagaaaga aagaaaacca
     3061 ggagtgaaaa ttagaaaagg aaaggaaagg aaaaaaagaa aaatctgaaa atatataaaa
     3121 aaaaattgtt tcgttggcaa taaatcttgg tgagaacagc gaccgaaagc aaataagaac
     3181 aaaatatgag tgtattacgt tgaacaacta attaacgtgt gtgtatggat ctttttttct
     3241 tttttctctt taaccgacta taaacaacaa acatttttgg gcagtgcaca cactacttaa
     3301 tatacacagc ataaattaca cgattagaaa caaattagct tattaaaata acctaatcaa
     3361 accgaatatt ttatggtatt atgagtaaac tatataatat aaatagcaca cacccacaac
     3421 aacaacaaag gaaaactaaa aggttttttc tttttgaaaa gatcgttttc tttattattc
     3481 tctagttttg acggcggccg cagtgattag acttagtccg ttcaaatcaa gcacaactct
     3541 gttcattgtt tcaacaagaa ttaattcaaa aacaggttcg gtgcataatt tgcaaaaaaa
     3601 tattgcagct tctgtggctc gaacacagta cctccagatt tcaggtttga aatacttcag
     3661 tctgacgctc tcccagatga gctaaagctg caataagaaa acccacgccg ggattcgaac
     3721 ccggaatcct ttgattagaa gtcaaaagcg ataaccattt cgccacgcag gcctacttga
     3781 tgggtttgta aatggtctac tttttcagac ctaacagaaa ttttaatgaa agtcatattc
     3841 ttatacaata aaactgtgtc ataaaagcag atattcgact ttcgtagatt atataggacc
     3901 caagaactaa aatttaatgc catattatgc atttttaatc tgtaaaagtg ttgtttccaa
     3961 cctatcacaa gtacgttctt gtaacttgtg tttgtagggt tgcaaatgaa tcataacaac
     4021 atctcaacag aacatgtata gcaaagctta gtataaaatc agtgttttga gaggcaatcc
     4081 aagaatgttt acatcaaagt ttcaataaat atcgaccgaa actgaaaatc tttttaggtt
     4141 attgttcact tttttgtaaa tatttaaaca ttttttggac ctaaaaaaat acaaacacca
     4201 attacgtacc aagaagcatc taatcaactc ccagatcacc actatacatt taaaagtcat
     4261 tggtcaataa ctatactcga gtattgcctc atcaaagaaa caatcaaata ttatagatac
     4321 tcactccatc acgtgataat ttcactggta tggaaaagtg gaaaatttta taaaaaaaaa
     4381 tttgatgcct ttggcatagc tgaaacttcg gcccaatagg attggagaat atgttttcgc
     4441 agcgttctta caattaaatt gtggtggaag ttcgagactt gcgtaaacta tttttaattt
     4501 ggatccgcaa caatcatacg acctaatgtt ttagagctag aaatagcaag ttaaaataag
     4561 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctcgagtttt
     4621 tttatcgagt gtttaaggat aatgataact gaagagaaga attagttttt tcaagtgtat
     4681 aatatagttt ctctctatta ccttttccaa taatagcatt ttaagttttc tattttattt
     4741 tgtataaaaa acataatgaa aaatacgtat aagtaatata aatgagtgtg ggattaagtg
     4801 aatacgagat gttgtagtga taatagggga aactctttgg cgaaactaca agagagagtg
     4861 atgtgctaat aatgaacgaa gaaatatgtg atttttgtat gaaatttgca attattctga
     4921 ttgaatttgg gtacttgaca ttgaatccag aacgactata caaatgtgct actttgtcaa
     4981 aatatccttt ttgagaatcg gcatatttat ggccctgaat atcgactacc acattccttt
     5041 tacaacacta cgtaaccttt tgagaaagta caagtgaaag aagtatagaa ttcagtgttt
     5101 agtttaacgt aagtattact gtggaatgct ttcttcgcga cacaagcaac ttgtacctgc
     5161 acccttcaca caatttattt cctaaaacta ctccagtgcg aaaacaatag tgctaaatat
     5221 gatgatgaga gaattcttaa cgaacggagt aggaatgtac atactatcac tagtttccaa
     5281 ataacaaaaa taaaaaaaaa aataacatgg aacttgtatt gctaaataaa ttactagatt
     5341 ttataagcaa taaaaagaat ttgaaaagga tgcttcatca caactaatag tttagtttct
     5401 ttacttctcc cctgtttact gggttatttt atttagatta tgctaatata attttttaat
     5461 acaagaattt ttattttttt aatttatgtt gctgattgcc cctaaaattt caaattcctg
     5521 aaattccctg agtgacttga acccagacac acattcactc actcacacaa acaaatacac
     5581 aaaattagag aacctgaatt tcagattctc aaattccaaa acagcaaagc cgcggtggag
     5641 ctccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     5701 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     5761 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     5821 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5881 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     5941 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     6001 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     6061 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     6121 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     6181 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     6241 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     6301 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     6361 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     6421 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     6481 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     6541 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     6601 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     6661 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     6721 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6781 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6841 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6901 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6961 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     7021 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     7081 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     7141 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     7201 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     7261 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     7321 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     7381 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcaga
//
